Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes are widely used to model cardiac development and disease, but directed differentiation produces heterogeneous cellular states whose regulatory mechanisms remain incompletely defined. Here, we used simultaneous high-throughput ATAC and RNA Expression with sequencing (SHARE-seq) to profile gene expression and chromatin accessibility during human iPSC-derived cardiomyocyte differentiation. Weighted nearest neighbor integration resolved sequential developmental cell states, including iPSCs, mesodermal cells, progenitor populations, cardiomyocytes, and off-target, mixed, or partially differentiated populations. Peak-to-gene linkage analysis nominated cell state-associated putative enhancer-gene relationships, including a cardiac-related regulatory linkage at the MYH6/MYH7 locus. Pseudotime analysis revealed progressive remodeling of transcriptional and chromatin-associated regulatory programs. In silico perturbation modeling further prioritized transcription factors (TFs) predicted to influence differentiation trajectory progression, and gene regulatory network inference identified candidate TF-centered modules associated with cardiac lineage progression. Comparative pseudotime analysis of these modules showed that motif-containing regulatory element accessibility appeared to emerge earlier than, or in parallel with, TF expression and downstream target module activation. Together, these findings provide a single-cell multimodal resource and analytical framework for linking cellular identity, chromatin accessibility, putative enhancer-gene relationships, and candidate transcription factor regulatory programs during human cardiomyocyte differentiation.
Spermatozoa are increasingly recognized as carriers of non-genetic paternal information, rather than passive vehicles for the haploid genome. During spermatogenesis and post-testicular epididymal maturation, the paternal germline establishes a compact but functionally organized epigenome composed of DNA methylation, retained histones and histone modifications, chromatin-associated factors, and diverse small RNAs. These layers are environmentally responsive. In animal models, paternal diet, stress, toxicant exposure, and inflammatory or microbiome-related challenges can alter sperm DNA methylation, chromatin states, and small RNA cargo, with many changes mapping to loci involved in development, metabolism, and stress-response pathways. After fertilization, the paternal genome undergoes extensive epigenetic reprogramming; nevertheless, a subset of DNA methylation and chromatin features can resist erasure or be functionally relayed, while sperm-derived RNAs can influence early embryonic gene expression. Recent work, including studies of diet-induced sperm mitochondrial tRNAs, further supports the concept that defined paternal exposures may be transmitted to the embryo through discrete RNA-mediated mechanisms. In humans, lifestyle and environmental exposures are associated with measurable sperm epigenomic variation and with offspring health outcomes, but most evidence remains observational and is vulnerable to confounding by genetics, maternal factors, and shared environments. A central challenge is therefore to connect specific sperm epigenetic alterations to molecular effects in the early embryo and to subsequent offspring phenotypes. Here, we review how paternal environmental information is encoded during spermatogenesis and epididymal maturation, reshaped by environmental exposures, and interpreted by the early embryo, emphasizing multilayer integration and the evidence required to move from association to mechanism.
Potassium channel tetramerization domain-containing protein 17 (KCTD17) is part of the BTB/POZ domain-containing KCTD protein family and serves as a multifunctional regulator of cellular homeostasis. As an adaptor protein associated with Cullin 3 (CUL3)-based E3 ubiquitin ligase complexes, KCTD17 plays a critical role in selective protein ubiquitination and proteasomal degradation, influencing the stability of various cellular proteins. This function links KCTD17 to numerous biological processes, including organelle dynamics, cell differentiation, intracellular signaling, metabolic regulation, stress responses, tissue remodeling, and disease progression. Notably, the effects of KCTD17 are highly context-dependent, varying with cell type, substrate availability, and pathological conditions. While recent studies have begun to uncover several pathways associated with KCTD17, its complete range of substrates, regulatory mechanisms, and physiological significance are still not fully understood. In this review, we summarize the current understanding of KCTD17's molecular functions and biological mechanisms, emphasizing its role as a proteostatic regulator. We also explore its potential relevance to human diseases and highlight key unresolved questions, such as substrate specificity, tissue-dependent functions, functional redundancy with related KCTD proteins, and therapeutic potential. Gaining a deeper understanding of KCTD17-dependent protein regulation may offer new insights into proteostasismediated cellular regulation and disease mechanisms.
Cellular identity is maintained by precise epigenetic regulation, but chronic stress and aging can disrupt this control, leading to mesenchymal drift (MD). Unlike the coordinated process of epithelial-mesenchymal transition (EMT), MD is a stochastic, lineage-independent phenomenon marked by increased epigenetic noise and the widespread activation of profibrotic gene programs. As originally defined, MD requires two components to co-occur: a compromised original cellular identity together with the acquisition or intensification of mesenchymal traits. Applying the MD framework to Alzheimer's disease (AD) provides a new perspective on its pathogenesis. In AD, brain cells do not merely survive or die due to protein toxicity; instead, they lose their specialized identities in an uncoordinated fashion. This transcriptomic drift has been reported across major cell types in the central nervous system: neural progenitor cells differentiate prematurely, mature neurons undergo transcriptomic dedifferentiation, endothelial cells develop features of vascular fibrosis, and glial cells lose their homeostatic functions. Direct enrichment of the MD signature in the AD brain, however, has so far been demonstrated only in astrocytes, oligodendrocytes, and pericytes, and, in our own reanalysis, in human-induced pluripotent stem cell (hiPSC)-derived neural progenitor cells carrying an APP mutation; we distinguish this evidence from observations documenting identity loss or a reactive state alone. We further caution that the novel cell populations identified in recent single-cell transcriptomic studies should not be assumed to represent genuinely new cell types: such clusters may equally reflect a blurring of existing cell lineages, or technical artifacts that produce the same appearance. A drifting transcriptomic profile also does not always signify a corresponding physiological change. These possibilities warrant careful attention, and we therefore emphasize the critical need to integrate transcriptomic data with in vivo functional and morphological validation. This integration is essential to determine whether these identity shifts contribute to AD progression or merely reflect underlying epigenetic instability.
MicroRNA-31 (miR-31) acts as a tumor suppressor and is downregulated in breast cancer, but the mechanism responsible for its reduced maturation remains unclear. To investigate this, we examined the expression patterns of pri- and mature miR-31 across multiple human tumor cell lines. Quantitative PCR revealed that while pri-miR-31 was abundantly expressed, mature miR-31 levels were selectively decreased in breast cancer cells, indicating a block at the post-transcriptional processing step. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) restored miR-31 expression while concurrently suppressing Galectin-3, suggesting a potential regulatory link between the two molecules. RNA electrophoretic mobility shift assays (EMSA) demonstrated direct binding of recombinant Galectin-3 to in vitro-transcribed pre-miR-31. Protein-RNA docking analysis further supported this interaction, identifying the CUGGG motif (C35-G39) within pre-miR-31 as the key contact region interacting with the carbohydrate recognition domain (CRD) of Galectin-3. Functional analyses in MCF7 cells, which exhibit the highest endogenous Galectin-3 levels, showed that Galectin-3 knockdown markedly increased mature miR-31 expression without altering pri-miR-31 levels, confirming regulation at the processing stage. TaqMan™ microRNA profiling revealed that Galectin-3 depletion selectively altered a subset of miRNAs, including miR-31, suggesting partial remodeling of the cellular miRNA landscape. Collectively, our findings identify Galectin-3 as a negative regulator of miR-31 maturation in breast cancer cells and suggest a novel role for this lectin in post-transcriptional control of microRNA biogenesis.
Hepatocellular carcinoma (HCC) displays significant molecular heterogeneity that clinical staging alone does not fully account for when assessing the risk of recurrence following curative treatment. To address this, we developed a multi-cohort survival modeling framework that translates bulk RNA-seq profiles into biologically meaningful tumor lineage scores. These scores are then used to stratify disease-free survival (DFS). Our study included 1,059 patients from four independent cohorts, where tumor immunogenic and proliferative lineage scores satisfied the proportional hazards assumptions and were integrated into a cohort-stratified Cox model. Each patient received a linear predictor (LP), and predefined cutpoints were established to categorize them into actionable risk groups. These groups exhibited consistent DFS separation in both the training (70%) and independent test (30%) sets. In ROC analyses, the LP demonstrated moderate overall discrimination (AUC 0.629, 95% CI 0.595-0.662). It showed higher discrimination for early recurrence (＜1 year; AUC 0.653, 95% CI 0.591-0.715) and lower discrimination for later recurrence (3-5 years; AUC 0.576, 95% CI 0.473-0.678). This approach establishes a biologically informed framework for stratifying recurrence risk based on RNA-seq data, potentially enhancing riskadapted surveillance and postoperative management for HCC.
T cell-based immunotherapies have transformed the treatment of hematological malignancies, but their efficacy in solid tumors remains inconsistent. Unlike blood cancers, solid tumors present multiple barriers that impede T cell infiltration, metabolic fitness, antigen recognition, and long-term persistence. These barriers include structural exclusion by the stroma, tumor-driven metabolic competition, antigen plasticity, and the progressive epigenetic fixation of T cell exhaustion states. This review integrates our current understanding of T celldirected therapeutic approaches and examines the tumorintrinsic and microenvironmental mechanisms that limit their activity in solid malignancies. We discuss how chronic stress signaling, altered nutrient availability, glycan-mediated epitope masking, and transcriptional reprogramming collectively destabilize therapeutic T cell function. Finally, we evaluate emerging strategies designed to remodel the tumor niche, diversify antigen targeting, and enhance T cell metabolic and epigenetic resilience. Thus, developing a mechanistic framework that combines intrinsic T cell reprogramming with adaptation to the tumor context will be crucial for extending durable T cell-mediated immunity to solid cancers.
Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide and is associated with a poor prognosis, even when diagnosed in its early stages. Therefore, identifying novel HCC targets for therapeutic drug development is critically needed. Our proteomic analysis identified Glucose-regulated protein 78 (GRP78) as a potential new target, showing enrichment in HCC spheroids. We observed that post-surgery HCC patients with elevated GRP78 expression in non-tumor tissues had an unfavorable prognosis. Inhibition of GRP78 expression suppressed cell proliferation and reduced viability by controlling the DNA damage response in HCC cells. GRP78 expression peaked during the G2/M phase of mitosis and was predominantly localized to the cytoskeleton. However, its expression decreased as the cell cycle progressed from the G2/M to the G1 phase in HCCs. Overexpression of GRP78 in HCC cells increased the interaction between PLK1 and GRP78, leading to the stabilization of PLK1 in the midbody during cytokinesis. This stabilization resulted in the failure of cells to exit mitosis, producing multinucleated HCC cells. These findings reveal novel functional roles for GRP78 in disrupting mitotic activity through the stabilization of PLK1 at the midbody, thereby fostering polyploidy development in HCC. This suggests that inhibiting GRP78 is a promising approach for enhancing HCC therapy. [BMB Reports 2026; 59(8): 398-406].
Alzheimer's disease (AD) is marked by progressive cognitive decline and retinal abnormalities, including degeneration of the retinal pigment epithelium (RPE). In models of AD, the accumulation of amyloid-β (Aβ) peptides triggers chronic inflammation and activates the immunoproteasome (iP), primarily driven by tumor necrosis factor-alpha (TNFα) from glial cells. Although pharmacological inhibition of iP has demonstrated protective effects against retinal damage, the precise molecular mechanisms involved remain unclear. In this study, we reveal that Aβ stimulation initiates a signaling cascade from glia to RPE, mediated by TNFα, which in turn promotes iP activation and upregulation of the NLRP3 inflammasome in RPE cells. Aβ exposure significantly increased TNFα secretion in Müller glia, whereas RPE cells exhibited only a minimal intrinsic response to TNFα secretion in response to Aβ. Inhibition of iP reduced TNFα release from glial cells. Additionally, neutralizing gliaderived TNFα decreased iP activity and mitigated epithelialmesenchymal transition (EMT) in RPE cells, highlighting TNFα's crucial role in glial-induced retinal inflammation. These findings position glia-derived TNFα as a central factor in Aβ-induced RPE degeneration through the TNFα-iP-NLRP3 pathway, supporting the use of iP inhibition as a potential therapeutic approach to maintain retinal integrity in AD.
Hair loss is a psychologically debilitating condition affecting social interactions. Despite extensive research, current interventions are transient and provide limited efficacy. Emerging evidence highlights that oxidative stress, resulting from mitochondrial dysfunction, impairs hair growth and disrupts hair cycle regulation. Urolithin A (UA), known to enhance mitochondrial function via mitophagy activation, has not yet been studied for its protective effects against hair loss. Here, we investigate whether UA protects against oxidative stress and promotes hair growth by restoring mitochondrial function in human dermal papilla cells (hDPCs) and clinical cases. hDPCs were subjected to H2O2 to induce oxidative stress and treated with various UA concentrations. Protective effects were assessed via mitochondrial function, morphology, apoptosis assays and Wnt/β-catenin signaling activity. Clinical evaluation involved measuring hair root volume and shedding rates following topical UA application. UA treatment improved mitochondrial function, reduced cell death under oxidative stress conditions, and activated Wnt/β-catenin signaling by increasing Wnt and β-catenin expression in hDPCs. Clinically, UA application led to increased hair root volume and decreased hair shedding. These dual effects on mitochondrial function and Wnt/β-catenin signaling highlight the potential of UA as a novel intervention for hair loss management.
Epigallocatechin gallate (EGCG), a major polyphenol in green tea, exhibits anti-obesity effects, but its precise mechanism remains to be fully elucidated. In this study, we found that EGCG suppresses the adipocyte differentiation of 3T3-L1 cells, which was achieved with its treatment limited to the first two days post-differentiation induction. EGCG inhibited the tetranectin (TN)-mediated increase in ERK and AKT phosphorylation. EGCG efficiently removed TN in the differentiation medium, suggesting TN as a direct target. Furthermore, TN was found to undergo endocytosis during early differentiation, which was attenuated by EGCG, indicating a potential intracellular role for TN. Collectively, these findings demonstrate that EGCG suppresses adipogenesis by reducing TN availability in the medium and inhibiting its subsequent endocytosis, thereby modulating the ERK and AKT signaling pathways.
Prime editing is a next-generation genome editing technology that enables precise base substitutions, insertions, and deletions at target genomic loci without inducing double-strand breaks or requiring exogenous donor DNA. This system operates through a fusion protein composed of Cas9 nickase and reverse transcriptase together with prime editing guide RNA; it has emerged as a precise genome editing platform that overcomes the limitations of conventional double-strand break-inducing CRISPR-Cas9 systems. Since first reported in 2019, diverse methodological improvements from PE1 to PE7 have been achieved, leading to rapid advances in editing efficiency, expansion of the editable target range, correction of large genomic regions, and development of in vivo delivery technologies. In this review, we comprehensively discuss the fundamental working mechanism of prime editing, its methodological evolution, recent expansion strategies, and delivery platforms for therapeutic applications, and provide perspectives for future development. [BMB Reports 2026; 59(6): 313-320].
Targeting the behavior of myeloid-derived suppressor cells (MDSCs), either by decreasing their population or inhibiting their immunosuppressive activity, has become a promising approach in breast cancer therapy. As pivotal mediators within the tumor microenvironment (TME), MDSCs facilitate tumor progression via diverse mechanisms, including T-cell suppression, epithelial-mesenchymal transition (EMT), and the preservation of cancer cell stemness. EK-16005, a novel 2-anilinopyrimidine derivative, has shown robust inhibitory activity against breast cancer cells. In this investigation, we examined the impact of EK-16005 on the reciprocal interactions between MDSCs and cancer cells that modulate cytokine production and signaling pathway activation. EK-16005 downregulated the secretion of critical cytokines such as G-CSF, VEGF, and CXCL1/2, leading to blockade of STAT3 and Notch signaling cascades in vitro and in vivo. As a result, EK-16005 decreased MDSC expansion, tumor growth, and cancer stemness properties. Collectively, these results establish EK-16005 as a potential therapeutic candidate for interrupting MDSC-mediated tumor progression and improving outcomes in breast cancer treatment.
Colorectal cancer (CRC) poses major clinical challenges, and deeper molecular and immunological insights are essential for precision oncology. We integrated RNA sequencing (RNA-seq) and clinical data from a Korean CRC cohort to profile tumor biology and immune heterogeneity. Differential expression analysis revealed 1,652 upregulated and 1,372 downregulated genes in tumors, with enrichment of DNA damage response (DDR), WNT signaling, and extracellular matrix (ECM)- receptor pathways, whereas normal tissues were enriched for metabolic processes. Consensus Molecular Subtype (CMS) classification identified canonical subtype-specific features, with unclassified tumors resembling CMS2. Immune phenotyping stratified tumors into hot, intermediate, and cold groups, showing distinct associations with microsatellite instability (MSI) and CMS subtypes. Tumor Immune Dysfunction and Exclusion (TIDE) analysis predicted responders across all phenotypes, unexpectedly including intermediate and cold tumors. Notably, interferon-STAT3-PIM1 signaling characterized non-responders, implicating oncogenic drivers of immune resistance. Immunehot tumors displayed strong antigen presentation (HLA-C, B2M) and apoptosis (FAS) signatures, supporting sensitivity to PD-1 blockade with potential benefit from apoptosis-targeting agents. Intermediate tumors showed partial CD8＋ T cell activation, suggesting combinatorial strategies such as CTLA-4 blockade or oncolytic priming. Cold tumors with IRF1 expression exhibited latent immune potential, indicating opportunities for epigenetic modulation plus checkpoint inhibition. These findings highlight the immune transcriptomic diversity of CRC and demonstrate that immune phenotype-based stratification provides actionable insights beyond MSI or CMS classification. This framework may guide personalized immunotherapy strategies for Korean CRC patients and inform future integration with spatial transcriptomic profiling.
The specific roles of single-nucleotide polymorphisms and structural variants (SVs) in Korean patients with inflammatory bowel disease (IBD) susceptibility remain unclear; hence this study aimed to evaluate polygenic risk scores (PRS) for IBD and identify IBD-associated SVs in a Korean IBD cohort. Whole genome sequencing data from 75 Korean patients with IBD were analyzed, and compared with individuals from the general Korean population cohort. The PRS models significantly distinguished patients with IBD from the general Korean population, confirming the predictive power of PRS in Korean patients with IBD. SV analysis revealed IBD-associated deletions, with candidate loci including PLA2R1, PTPN2, LINC00484, CCND3, COMMD7, and ERAP2. Of these, DEL5948 in ERAP2 overlapped multiple IBD-associated SNP loci and disrupted key regulatory and coding regions, potentially influencing immune regulation. This study provides a comprehensive genome-wide analysis of PRS and SVs in Korean patients with IBD, highlighting novel genetic factors in IBD pathogenesis.
Metabolic dysfunction-associated steatohepatitis (MASH) is a significant global health issue and a leading cause of liver fibrosis, with no effective pharmacological treatments available. Conventional 2D cell cultures and animal models do not fully recapitulate the complex cell-cell interactions and multicellular microenvironment that drive disease progression in humans, which limits their ability to predict therapeutic efficacy accurately. The recent introduction of the FDA Modernization Act 2.0 has paved the way for the use of human-relevant New Approach Methodologies (NAMs) in drug discovery and toxicity testing. This review focuses on recent advancements in NAMs that model MASH-associated liver fibrosis, such as human liver spheroids, iPSC-derived liver organoids, precision-cut liver slices, 3D bioprinting, and liver-on-a-chip systems. By mimicking multicellular interactions and the liver microenvironment, NAMs offer a valuable platform for mechanistic studies and drug screening. Assessing their strengths and limitations can lead to deeper mechanistic insights and expedite the development of new therapeutics for MASH-associated liver fibrosis.
Hepcidin (encoded by the HAMP gene), produced primarily by hepatocytes, is the master regulator of systemic iron homeostasis. Its dysregulation contributes to various iron-related metabolic disorders. Cereblon (CRBN) has been implicated in metabolic regulation, while estrogen-related receptor gamma (ESRRG) is known to govern energy homeostasis and mitochondrial function. In this study, we demonstrate a novel CRBN-ESRRG signaling pathway that mediates endoplasmic reticulum (ER) stress-induced hepatic HAMP expression. In mice and primary hepatocytes exposed to tunicamycininduced ER stress, gene expression and biochemical analyses revealed significant increases in the transcript levels of hepatic Crbn, Esrrg, and Hamp. Correspondingly, hepcidin protein levels were elevated, accompanied by reduced serum iron levels and increased cellular iron levels, consistent with hepcidinmediated regulation of iron distribution. Overexpression of Crbn enhanced ESRRG expression and increased hepatic hepcidin production, while knockdown of either Crbn or Esrrg attenuated this response. Chromatin immunoprecipitation assays demonstrated enhanced recruitment of ESRRG to the Hamp promoter. Collectively, these findings identify a CRBN-ESRRG regulatory axis that drives hepatic HAMP expression under ER stress and suggest a potential therapeutic target for ER stress-associated metabolic and iron disorders. [BMB Reports 2026; 59(5): 291-298].
Obesity is a major global health crisis, yet the molecular mechanisms underlying adult-onset metabolic dysfunction remain incompletely understood. The tubby mouse is a foundational genetic model of maturity-onset obesity; however, the specific tissues and cell populations responsible for its metabolic phenotype have long remained elusive. Here, we demonstrate that the loss of tubby disrupts the coordinated regulation of energy intake and expenditure, leading to a sustained positive energy balance. Using cell-type-specific genetic tools, we identified MC4R-expressing and VGLUT2-expressing neurons as essential sites of tubby function. We found that tubby acts through the combined contribution of these neuronal populations, as selective deletion in either MC4R or VGLUT2 neurons is sufficient to phenocopy key features of the global Tub mutant. Together, these findings establish tubby as a central neuronal regulator of systemic energy homeostasis and define an excitatory MC4R-VGLUT2 circuit that governs feeding behavior and metabolic output. [BMB Reports 2026; 59(5): 293-290].
Plant-derived extracellular vesicles (PDEVs) have attracted increasing attention as bioactive nanostructures with emerging applications in medicine, functional foods, and cosmetics. However, unlike mammalian extracellular vesicle systems, the recovery of PDEVs is fundamentally constrained by plant-specific structural features, particularly the rigid cell wall and the limited accessibility of vesicles within plant tissues. In plant systems, extracellular vesicles are thought to be retained, at least in part, within the apoplastic space between the plasma membrane and the cell wall, which restricts the direct application of conventional animal cell-based isolation workflows. Conventional extraction approaches based on mechanical disruption enable bulk recovery of vesicle-containing material but are frequently associated with intracellular contamination, cell wall-derived debris, increased extract viscosity, and poor process reproducibility. To address these limitations, recent studies have explored enzyme-based extraction strategies that partially relax the plant cell wall to facilitate the selective release of apoplastic vesicles while reducing nonspecific contamination. In this mini-review, we summarize recent advances in enzyme-based extraction strategies for PDEVs, with particular emphasis on their structural rationale, methodological advantages over mechanical disruption, and associated limitations. We further discuss key challenges in quantitative evaluation, purity assessment, and reporting standardization, highlighting the need for methodologically rigorous frameworks to enable reproducible isolation and reliable interpretation of PDEV-associated biological activities.
Cellular senescence is an irreversible program of cell-cycle arrest that accumulates with age, contributing to chronic inflammation and various age-related diseases. A key feature of senescence paradigms is mitochondrial dysfunction, which involves not just a single defect but a series of coordinated changes in bioenergetics, redox homeostasis, mitochondrial quality control, and organelle interaction. Senescent cells often display a "quantity-quality imbalance" in their mitochondria: while the mitochondrial mass may increase, their efficiency in oxidative phosphorylation decreases, leading to a destabilized membrane potential (ΔΨm) and elevated levels of mitochondrial reactive oxygen species (mtROS). These interrelated changes can exacerbate senescence through persistent stress signaling, impaired turnover of damaged mitochondrial components, and alterations in organelle contacts, such as those between endoplasmic reticulum (ER) and mitochondria, and between mitochondria and lysosomes. Given that these phenotypes differ depending on cell type, triggering factors, and timing, no single assay can adequately define senescenceassociated mitochondrial dysfunction. In this review, we present practical, complementary strategies that include extracellular flux-based respiration profiling, ATP output measurement, ΔΨm and ROS assessments, flux-based mitophagy reporters, quantitative network imaging, and contact-site assays. We propose minimal assay bundles that allow for a thorough multidimensional analysis. By establishing standardized, orthogonal measures of mitochondrial quantity and quality, we aim to enhance mechanistic understanding and facilitate the rational evaluation of mitochondria-targeted senolytic and senomorphic therapies. [BMB Reports 2026; 59(3): 177-186].
